Papain-catalyzed aldol reaction for the synthesis of trifluoromethyl carbinol derivatives

Article abstract of DOI:10.1016/j.molcatb.2016.05.014

Papain from Carica papaya demonstrated catalytic promiscuity was first discovered to catalyze the synthesis of trifluoromethyl carbinol derivatives via aldol reaction between α,α,α-trifluoromethyl ketones and aliphatic ketones in a mixed solvent of DMF an

Full text of DOI:10.1016/j.molcatb.2016.05.014

Accepted Manuscript
Title: Papain-catalyzed aldol reaction for the synthesis of
trifluoromethyl carbinol derivatives
Author: Jun-Tao Guo Yang Xiang Zhi Guan Yan-Hong He
PII:
S1381-1177(16)30091-1
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2016.05.014
MOLCAB 3375
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Journal of Molecular Catalysis B: Enzymatic
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Revised date:
Accepted date:
1-3-2016
19-5-2016
31-5-2016
Please cite this article as: Jun-Tao Guo, Yang Xiang, Zhi Guan, Yan-Hong He, Papain-
catalyzed aldol reaction for the synthesis of trifluoromethyl carbinol derivatives, Journal
of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2016.05.014
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Papain-catalyzed aldol reaction for the synthesis of
trifluoromethyl carbinol derivatives
Jun-Tao Guo, Yang Xiang, Zhi Guan*, Yan-Hong He*
Key Laboratory of Applied Chemistry of Chongqing Municipality, School of
Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR
China
E-mails: guanzhi@swu.edu.cn (Z. Guan); heyh@swu.edu.cn (Y.-H. He)

Graphical Abstract
Papain was used for the first time as a biocatalyst in aldolreaction between
α,α,α-trifluoromethyl ketones and aliphatic ketones.
Highlights




Papain-catalyzed aldol reaction was described.
17 examples of aldol products were obtained by this reaction.
The novel reaction also expands the field of organic synthesis.
This work promotes the development of enzyme catalytic promiscuity.
Abstract: Papain from Carica papaya demonstrated catalytic promiscuity was first
discovered to catalyze the synthesis of trifluoromethyl carbinol derivatives via aldol
reaction between α,α,α-trifluoromethyl ketones and aliphatic ketones in a mixed
solvent of DMF and water. The best results of the corresponding aldol products with
up to 99% yield and 30% ee were achieved.
Key words: papain; aldol reaction; trifluoromethyl carbinol derivatives; enzyme
catalytic promiscuity

1. Introduction
The asymmetric aldol reaction serves as one of the most powerful carbon-carbon
bond forming methods, providing access to β-hydroxycarbonyl compounds in an
enantioselective fashion [1]. Ever since List [2], Barbas [3, 4], and MacMillan [5]
groups reported their pioneering works on organocatalyzed enantioselective aldol
reactions, a lot of efforts have been made on the organocatalysis and remarkable
progress has been achieved [6-14]. However, the use of ketones as electrophilic
partners, which provides access to chiral tertiary alcohols, still remains challenging
due to their poor reactivity and difficulty in differentiating the two faces of the
carbonyl moiety [15-17].
Organofluorine compounds are brought to the forefront as pharmaceuticals,
agrochemicals, functional materials, or catalysts [18-21]. More than 20% of medicinal
and agrochemical products consist of one or more fluorine atoms [22-24]. In the big
family, α-trifluoromethyl tertiary alcohol compounds play an important role due to
their bioactivities and stereoelectronic properties [25]. On the other hand, these
compounds are important synthetic intermediates; they can be converted to α-amino
acids, α-hydroxyl acids, oxiranes, and α-fluoro acids [26-31].
There is no doubt that the cross-aldol reactions of trifluoromethyl ketones with
ketones could be considered as one of the most useful approaches for the construction
of chiral trifluoromethyl tertiary alcohols. However, until recently, only a few
cross-aldol reactions of trifluoromethyl ketones with ketones have been reported
[32-43]. In 2005, Zhang and co-workers reported the first example of a
proline-catalyzed asymmetric aldol addition of aryltrifluoromethyl ketones to methyl
ketones, affording β-trifluoromethyl-β-hydroxyl ketones in satisfactory yields, with
moderate enantioselectivity of up to 64% ee [32]. After that, other successful catalytic
reaction systems with improved enantioselectivities have been disclosed by the Liu
[33, 34], Yuan [35], Nakamura [36], Berkessel [37], Kokotos [38], Song [42] and
Tanaka [43] groups with the use of organocatalysts.
Enzyme catalysis, as efficient and green biotransformation tools in organic
synthesis, show immense advantages such as mild reaction conditions, simple

separation, good selectivity, high yields, etc [44-46]. Nowadays, a growing number of
enzymes have been found to be capable to catalyze synthetic reactions which vary
from their natural roles [47-49], and this phenomenon is considered as enzymatic
promiscuity. In recent years, enzymatic promiscuity has been paid much attention by
chemists and biochemists [50-52]. Some enzymes have exhibited their promiscuity
through catalyzing the formation of C-C and C-heteroatom bonds [53, 54], such as the
aldol reactions [55-57], Markovnikov additions [58], Michael additions [59], Mannich
reactions [60], the asymmetric synthesis of α-aminonitrile amides [61],
multi-component cascade or domino reactions, etc [62, 63]. Inspired by the pioneers’
work, we wondered if it was possible to develop a novel and highly efficient catalytic
system for the asymmetric aldol reactions of trifluoromethyl ketones with aliphatic
ketones. After a wide screening of different hydrolases, we found that the direct
asymmetric aldol reaction for the synthesis of α-trifluoromethyl tertiary alcohol
derivatives could be catalyzed by papain (EC 3. 4. 22. 2), the most abundant cysteine
proteinase, in the latex of the unripe fruit of Carica papaya [64]. Wide substrate
scopes were also investigated. This finding provides a novel example of enzymatic
promiscuity.
2. Results and discussion
Initial studies were undertaken using α,α,α-trifluoromethyl-phenylethanone and
acetone as the model reaction. To confirm the specific catalytic effect of papain on the
model reaction, some control experiments were performed (Table 1). These control
experiments were carried out under the optimized conditions [details of the
optimizations were described hereafter in the paper ( from Table 2 to Table 5)]. In the
absence of papain, the reaction only gave the product in 5% yield (Table 1, entry 1).
The reaction with papain provided the product in an excellent yield of 99% with 30%
ee (Table 1, entry 2), indicating that papain preparation has a catalytic effect on the
aldol reaction. Because the active site of papain consists of Cys, His and Asn residues,
to further prove enzymatic activity, cysteine protease inhibitor methyl
(methylsulfinyl)methyl sulfide (MMTS) [65] was used to denature papain by

irreversible covalent modification, and extra MMTS was then removed by dialysis
against deionized water. The reaction with MMTS pretreated papain only gave the
product in a low yield of 29% with 13% ee (Table 1, entry 4). Meanwhile, as a
comparison, papain without modification was dialyzed against deionized water and
then used to catalyze the model reaction, which gave a good yield of 73% with 15%
ee (Table 1, entry 3). The results indicated that papain still maintained most activity
even undergoing the dialysis process; the chemical modification of papain by MMTS
indeed caused a serious loss of activity (Table 1, entries 3 and 4). The above
experiments suggested that cysteine is crucial for the reaction. To further confirm the
catalysis of papain on the model reaction, phenylmethylsulfonyl fluoride (PMSF), an
irreversible inhibitor of serine/cysteine protease which inhibits most cysteine
proteases like papain, was then used to denature papain, and the reaction with
PMSF-pretreated papain only gave the product in a low yield of 18% with 1% ee
(Table 1, entry 6) which indicated that PMSF strongly inhibited enzyme activity in
the aldol reaction. Furthermore, diethyl pyrocarbonate (DEPC), a known modifier of
the imidazole groups of histidine [66], was used to pretreat papain. And the
DEPC-pretreated papain almost completely lost its catalytic activity on the aldol
reaction (Table 1, entry 8). In addition, because metal ions can combine with enzymes
irreversibly and disrupt the three-dimensional structure of enzymes, metal ions Ag+
and Cu2+ was used to pretreat papain. It can be seen that the metal-pretreated papain
almost completely lost its catalytic activity on the aldol reaction (Table 1, entries 10
and 12). To exclude the effect of MMTS, PMSF, DEPC, Ag+ and Cu2+ on the model
reaction, blank reactions were performed with the same amounts of the above
mentioned inhibitors which only gave trace amounts of the product, respectively
(Table 1, entries 5, 7, 9, 11 and 13). It was indicated that these inhibitors alone did not
have noticeable effects on the model reaction. Above control experiments suggested
that the native structure of papain is responsible for the aldol reaction and the catalysis
may take place in the active site of enzyme.
To better understand the relationship between natural and promiscuous activities
of papain, an enzymatic assay of papain on the hydrolysis of

N-benzoyl-L-argininethylester (BAEE) as natural activity was performed. The natural
activity of native and inhibitor-pretreated papain was measured. The natural activity
of untreated papain was 3.20 U·mg-1, which had the best catalytic activity on the aldol
reaction (Table 1, entry 2). After dialysis, both the natural and unnatural activities
decreased slightly (Table 1, entry 3). However, after pretreated by MMTS, PMSF,
Ag+ and Cu2+, both the natural and unnatural activities of the papain sharply decreased
(Table 1, entries 4, 6, 10 and 12). The results demonstrated that both the natural and
promiscuous activities of the enzyme were inhibited by the above mentioned
inhibitors. Based on the above experiments, it can be inferred that the natural active
center of papain was also responsible for its activity on the aldol reaction.
Since medium is one of the most important factors influencing the enzymatic
reactions, it is necessary to investigate the effect of different solvents on the
papain-catalyzed aldol reaction. Some solvents with different log P values were
screened and the results are shown in Table 2. In general, the reactions in solvents
with log P values between -1.30 and 0.28, such as DMSO, DMF, isopropanol and
acetone gave products in high yields of 97-99% (Table 2, entries 1-4). Relatively
lower yields were obtained in the solvents with log P values between 0.49 and 3.20,
such as THF, MTBE, 1,2-diCl-ethane, butyl acetate, toluene and cyclohexane (Table
2, entries 6, 8-10, 12 and 13). However, exceptions were observed for the reactions
with MeCN and 1,4-dioxane, in which low yields were received even though their log
P values are -0.33 and -1.10, respectively (Table 2, entries 7 and 11). The model
reaction under solvent-free conditions gave a yield of 81% (Table 2, entry 5). The
results clearly indicated that the reaction yield was significantly influenced by the
reaction media. Unfortunately, no satisfactory enantioselectivity was achieved in all
the tested solvents. Papain showed the relatively good stereoselectivity of 27% ee in
DMF with an excellent yield of 97% (Table 2, entry 4). Thus, DMF was chosen as the
optimum solvent for the papain-catalyzed aldol reaction. The absolute configuration
of the product was assigned by comparing with the known chiral HPLC analysis [38,

42].
The existence of water is crucial for the enzyme-catalyzed reactions in organic
solvents, as it influences the activity, stability of the enzymes and, probably, their
conformational flexibility. Thus, different water activities were screened for the
papain-catalyzed aldol reaction in the mixed solvent of DMF and water (Table 3).
Obviously, water activity had an influence on the yield of the model reaction. The
lowest yield of 66% with 23% ee was gained without adding water into the reaction
system (Table 3, entry 1). When water was added to DMF, better results were
obtained. Good yields of 92-98% were achieved with water activity ranged from 0.17
to 0.42 (Table 3, entries 2-5). When the water activity surpassed 0.47, the reaction
gave decreased yields (Table 3, entries 6 and 7). The best yield of 98% with 28% ee
was obtained with water activity value of 0.17 (Table 3, entry 2). Thus, water activity
value of 0.17 was selected for further studies.
After that, the effect of mole ratio of substrates on the model reaction was
investigated (The data is shown in supporting information). Among the performed
experiments, when the mole ratio of 1a/2a was 1:10, the product was obtained in an
excellent yield of 98% with the ee of 28%. Thus, the mole ratio of 1:10 (1a/2a) was
identified as the optimal ratio for further studies.
Then, in order to find out a suitable enzyme loading for the reaction, the effect of
papain loading on the model reaction was investigated (Table 4). When 32 U of
papain was used, the reaction only gave a moderate yield of 61% with 29% ee (Table
4, entry 1). Increasing the amount of papain to 96 U led to an excellent yield of 98%
with 29% ee (Table 4, entry 3). However, higher papain loading did not give better
results; the yield and ee almost kept constant (Table 4, entries 3 and 4). Thus, 96 U of
papain was chosen as the optimum enzyme loading for the reaction system of
trifluoromethyl ketone (0.6 mmol) and acetone (6.0 mmol) in a mixed solvent of
DMF (0.95 mL) and water (0.05 mL).

Since pH of the reaction system and volume of medium may also affect both
stereoselectivity and activity of a reaction. So, phosphate buffer saline (pH from 4.7 to
11.1) was used to replace the water in the reaction system (buffer/DMF = 5/95, v/v) to
obtain the optimum reaction conditions. Using buffer did not give obviously better
results. Thus, the deionized water was still used in the mixed medium for the reaction.
The volume of medium was also screened, and 1.25 mL of medium volume was
chosen as the optimum condition for further studies. The data is shown in supporting
information.
Temperature is a crucial factor affecting stability of enzymes, as well as
stereoselectivity and reaction rate of enzymatic reactions. Hence, it was essential to
investigate the influence of temperature on the papain-catalyzed aldol reaction. As
shown in Table 5, rising the temperature from 10 °C to 30 °C led to an increase of
both yield and ee (Table 5, entries 1-5). Further increasing the temperature to 50 °C,
the yield and ee changed to be steady (Table 5, entries 6-8). The model reaction
exhibited the best yield of 99% with 30% ee at 30 °C (Table 5, entry 5). The blank
reaction at 30 °C was also conducted, which only gave a low yield of 5% (Table 5,
entry 9). Thus, 30 °C was selected as the optimal temperature.
With these optimized conditions in hand, we next evaluated the applicability
scope of catalytic promiscuity embodied by papain. Some other substrates including
various trifluoroacetophenones and ketones were investigated to expand upon the
papain-catalyzed aldol reaction. The results are summarized in Table 6. To our delight,
the reactions between acetone and various aryltrifluoromethyl ketones with either
electron-rich or electron-deficient substituents gave products in excellent yields of
90-97% (Table 6, entries 1-6). However, when the phenyl ring was replaced by a
perfluorophenyl moiety, the yield dropped significantly. Perfluoroacetophenone
reacted with acetone gave an inferior yield of 42% (Table 6, entry 7). In the case of

perfluoroalkylphenyl ketones, the high yields of 95% and 96% were obtained (Table
6, entries 8 and 9). Even the CF3 group was replaced by the less electron-withdrawing
CF2Cl group an excellent yield of 99% was still received (Table 6, entry 10). The
substitutional-trifluoroacetone containing heteroatom reacting with acetone also
resulted in an excellent yield of 92% (Table 6, entry 11). Besides acetone, some other
aliphatic ketones were also used to react with trifluoroacetophenone. When
2-hydroxyacetone was utilized, a mixture of two diastereomers was obtained in
almost equal amounts with the moderate yield of 70% (Table 6, entry 12). The
reactions with 2-butanone and 2-pentanone gave acceptable yields of 72% and 75%,
respectively (Table 6, entries 13 and 14). Methoxyacetone could also react with
trifluoroacetophenone giving a yield of 56% (Table 6, entry 15). Moreover, this
papain-catalyzed aldol reaction could be expanded to alkyltrifluoromethyl ketones.
For instance, ethyl 4,4,4-trifluoroacetoacetate and ethyl trifluoropyruvate reacted with
acetone giving corresponding products in moderate yields of 58% and 67%,
respectively (Table 6, entries 16 and 17). Unfortunately, the enantioselectivities were
not satisfactory; the ee values observed were no more than 30% (data are not shown
in Table 6).
Furthermore, the kinetic parameters (Michaelis constant KM, catalytic constant
kcat and kcat/KM ) of the papain-catalyzed aldol reaction for each of the substrates in the
model reaction were determined (Table 7). It can be seen that the KM for each
substrate was quite high, showing that the affinity of papain to non-native substrates
was very poor. But the KM of acetone (2a) was lower than trifluoromethyl ketone (1a),
indicating that the affinity of papain with acetone is better than trifluoromethyl ketone.
Based on these results, we speculate that the papain-catalyzed aldol reaction of
acetone and trifluoromethyl ketone possibly takes place through the intermediate
enolate ion, generated from acetone mediated by enzyme (as shown in the possible
mechanism we proposed in Scheme 1). Meanwhile, both the kcat and kcat/KM were
very low, indicating that the reaction of papain for the promiscuous substrates was

very slow.
It is known that the active site residues Cys-25, His-159 and Asn-175 (papain
numbering) are involved in the papain catalytic process for hydrolysis. Cysteine and
histidine form the catalytic dyad in the active site of cysteine proteases. They are
thought to be present as a thiolate-imidazolium ion pair Cys 25-S-/His 159-Im+H in
the mature enzyme [67-69], which is stabilized by Asn-175 via keeping the imidazole
ring of His-159 in a favorable orientation [70]. A three-dimensional X-ray study at a
resolution of 2.8 Å has revealed that the single polypeptide chain of 212 residues is
folded into two distinct parts which are divided by a cleft. The active site cysteine
residue (Cys-25) is part of the L1 α-helix at the surface of the left domain, while the
histidine (His-159) is in a β sheet at the surface of the right domain of the enzyme [71,
72]. Our control experiments suggested that the native structure of papain is
responsible for the aldol reaction and the catalysis may take place in the active site of
enzyme. In 2003, Berglund et al. reported an aldol reaction catalyzed by Lipase B
Candida antarctica (CALB), in which the control reactions were run with covalently
inhibited wild-type CALB using the inhibitor methyl p-nitrophenyl
n-hexylphos-phonate. The inhibited enzyme did not show any lipase activity, and the
specific activity for the aldol reaction was as that of albumin. Then they mutated the
serine for a nonpolar residue alanine. The wild-type and the mutant enzymes were
used to catalyze the aldol reaction between aldehydes and ketones, the results
indicated that the mutant had better catalysis activity than the wild. They gave a
possible explanation that in the mutant enzyme the formation of hemiacetal by serine
attacking the substrate was avoided. And they proposed a mechanism, in which
histidine plays a key role for the catalysis of aldol reaction [73], and serine does not
directly participate in the catalytic process. Based on the mechanism of the lipase
CALB catalyzed aldol reaction proposed by Berglund et al. and our previous work on
the direct asymmetric aldol reaction of aldehydes with ketones catalyzed by
chymopapain, a papain-like cysteine protease [74], we hypothesized a mechanism for

the papain-catalyzed aldol reaction (Scheme 1). Firstly, the ketone is deprotonated by
His residue forming an enolate anion. Secondly, another substrate trifluoromethyl
ketone combines with the enolate anion and accepts a proton from imidazolium cation
leading to the aldol adduct. Eventually, the product is released from the active site.
3. Conclusion
In summary, we developed a novel synthetic methodology for the synthesis of
functionalized trifluoromethyl carbinol derivatives via enzyme-catalyzed aldol
reaction of trifluoromethyl ketones and aliphatic ketones in DMF/H2O system under
mild reaction conditions. Papain was used as an economically feasible and
environmentally friendly biocatalyst. Yields of up to 99% and ee of up to 30% were
achieved from readily available starting materials without using any additives.
Although the enantioselectivities were not satisfied, to the best of our knowledge, this
is the first example of enzyme-catalyzed aldol reaction between trifluoromethyl
ketones and aliphatic ketones. The use of ketones as electrophilic partners to provide
access to chiral tertiary alcohols is challenging due to their poor reactivity and
difficulty in differentiating the two faces of the carbonyl moiety. This work expands
the application of enzyme in organic synthesis, and may be a useful building block for
future study on papain catalytic promiscuity. Using the abundant papain as a catalyst
could be developed as a potentially valuable method which can be a complement to
the traditional chemical catalysis.
4. Experimental section
4.1 General procedure for the papain-catalyzed aldol reaction
To a 10 mL round bottom flask was added with trifluoromethyl ketone (0.6 mmol),
ketone (6.0 mmol), DMF (1.19 mL), deionized water (0.06 mL) and papain (96 U).
The flask was covered with a rubber stopper. The resultant mixture was stirred on a
magnetic stirrer for the specified time at 30 °C, and monitored by TLC [petroleum
ether/ethyl acetate (VPE/VEA = 3:1)]. The reactions were terminated by filtering the
enzyme. The ethyl acetate was employed to wash the residue on the filter paper to

assure that products obtained were all dissolved in the filtrate. The filtrate was washed
with water for three times, and then dried over anhydrous Na2SO4. The organic
solvent was then removed under reduced pressure. The crude products were purified
by silica gel column chromatography with petroleum ether/ethyl acetate as eluent
(VPE/VEA = 7:1 to 10:1).
4.2 Materials
Papain was purchased from Sigma-Aldrich, Shanghai, China. Papain from Carica
papaya (76220-25G, Lot# BCBD3116V, 3.6 units/mg solid), unit definition: one unit
corresponds to the amount of enzyme which hydrolyzes
1
µmol
N-benzoyl-L-argininethylester (BAEE) per minute at pH 6.2 and 25 °C. Unless
otherwise noted, all reagents were purchased from commercial suppliers and used
without further purification.
4.3 Analytical Methods
Reactions were monitored by thin-layer chromatography (TLC) with Haiyang
GF254 silica gel plates (Qingdao Haiyang chemical industry Co Ltd, Qingdao, China)
using UV light and vanillic aldehyde as visualizing agents. Flash column
chromatography was performed using 200-300 mesh silica gel at increased pressure.
1H NMR and 13C NMR spectra were recorded on Bruker-AM 600 (600 MHz) (Bruker
BioSpin AG Ltd., Beijing, China). Chemical shifts were reported in ppm from TMS
with the solvent resonance as the internal standard. Data were reported as follows:
chemical shifts (δ) in ppm, coupling constants (J) in Hz, and solvent (CDCl3).
High-resolution mass spectra were obtained by using ESI ionization sources (Varian
7.0 T FTICR-MS). Melting points were taken on a WPX-4 apparatus and were
uncorrected (Yice instrument equipment Co Ltd, Shanghai). The enantiomeric
excesses (ee) of aldol products were determined by chiral HPLC analysis performed
using Chiralpak AD-H, AS-H, Chiralcel OJ-H, OD-H columns (Daicel Chiral
Technologies CO., LTD.; Shanghai, China). Saturated vapor pressure was determined
by a DPCY-2C apparatus (company of NJUWH, Nanjing, China). Absolute

configurations of the products were determined by comparing with the known chiral
HPLC analysis. All the aldol products are known compounds except 3c and 3o (for
1
13
details about references, H NMR and C NMR of 3a-q, HRMS of 3c and 3o, and
HPLC charts of 3a, please see the supporting information)
Acknowledgements
This work was financially supported by the National Natural Science Foundation
of China (No. 21276211 and No. 21542014), and 2014 Chongqing Graduate
Education Reform Project (No.yjg143027).
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Scheme 1 Possible mechanism for the papain-catalyzed aldol reaction.

Table 1. Control experimentsa.
Natural
(U·mg-1)d
--
activity
Entry
Catalyst
Yield (%)b
Ee (%)c
1
No enzyme
5
0
3.20
2.30
0.47
--
2
Papain
99
30
15
13
--
1
3
Papaine
73
4
Papain (pretreated with 3.68 M MMTS)f
MMTS (0.9 mL)
29
5
Trace
18
6
Papain (pretreated with 0.43 M PMSF)g
0.28
--
7
PMSF (75 mg)
Trace
Trace
Trace
Trace
Trace
Trace
Trace
--
--
--
--
--
--
--
8
Papain (pretreated with 0.3 M DEPC)h
DEPC (43.3 μL)
--
--
9
10
11
12
13
Papain (pretreated with 0.25 M Ag+)i
0.19
--
AgNO3 (42.5 mg)
Papain (pretreated with 0.25 M Cu2+)j
CuSO4 (39.9 mg)
0.19
--
a Unless otherwise noted, reaction conditions: a mixture of trifluoromethyl ketone (0.6
mmol), acetone (6.0 mmol), papain (96 U), DMF (1.19 mL), deionized water (0.06
mL) at 30 °C for 72 h..
b Yield refers to the isolated yield.
c Ee was determined by HPLC analysis using a chiral column (OD-H).
d
Definition of enzyme activity (U·mg-1): one unit corresponds to the amount of
enzyme which hydrolyzes 1 µmol N-benzoyl-L-argininethylester (BAEE) per minute
at pH 6.2 and 25 °C.
e
Papain was dialyzed against deionized water and then water was removed by
lyophilization.
f
Papain (96 U) in MMTS solution (3.68 M) (0.9 mL MMTS in 1.5 mL MeCN) was
stirred at rt for 1 h, and then the 1 mL pH 5.0 acetic buffer was added. After stirred at
rt for 12 h the mixture was dialyzed against deionized water, and water was removed

by lyophilization.
g Papain (96 U) in PMSF solution (0.43M) (75 mg PMSF in 1.0 mL dried THF) was
stirred at rt for 12 h, and then organic solvent was removed under reduced pressure.
h To papain (96 U) in phosphate buffer solution (NaH2PO4-Na2HPO4, pH 8.04) (1 mL)
o
was added DEPC (0.3 mmol). The mixture was stirred at 37 C for 2 h, and then
water was removed by lyophilization.
i Papain (96 U) in Cu2+ solution (0.25 M) (39.9 mg CuSO4 in 1.0 mL deionized water)
was stirred at rt for 12 h, and then water was removed by lyophilization.
j Papain (96 U) in Ag+ solution (0.25 M) (42.5 mg AgNO3 in 1.0 mL deionized water)
was stirred at rt for 12 h, and then water was removed by lyophilization.

Table 2. Effect of solvents on the papain-catalyzed aldol reaction a.
Entry
1
Solvent
Log P
-1.30
0.28
-0.23
-1.00
--
Yield (%)b
99
99
Ee (% c
24
DMSO
2
Isopropanol
Acetone
24
3
99
97
81
67
60
56
42
41
40
36
31
23
4
DMF
27
5
Solvent-free
Cyclohexane
MeCN
25
6
3.20
-0.33
0.90
0.49
1.50
-1.10
2.50
1.70
28
7
27
8
MTBE
20
9
THF
23
10
11
12
13
1,2-DiCl-ethane
1,4-Dioxane
Toluene
17
19
16
Butyl acetate
17
a The reactions were conducted using trifluoromethyl ketone (0.6 mmol), acetone (6.0
mmol), papain (160 U), solvent (0.9 mL), deionized water (0.1 mL) at 25 °C for 72 h.
b Yield refers to the isolated yield.
c Ee was determined by HPLC analysis using a chiral column (OD-H).

Table 3. Effect of water activity on the papain-catalyzed aldol reaction a.
Entry Water / DMF (v/v)
Water activity
Yield (%)b
Ee (%)c
23
1
2
3
4
5
6
7
0/1.00
--
66
98
97
95
92
84
76
0.05/0.95
0.10/0.90
0.15/0.85
0.20/0.80
0.25/0.75
0.30/0.70
0.17
0.27
0.34
0.42
0.47
0.54
28
27
25
26
23
23
a The reaction conditions were as follows: trifluoromethyl ketone (0.6 mmol), acetone
(6.0 mmol), papain (160 U), DMF (1.00-0.70 mL), deionized water (0-0.30 mL) at
25 °C for 72 h.
b Yield refers to the isolated yield.
c Ee was determined by HPLC analysis using a chiral column (OD-H).

Table 4. Effect of enzyme loading on the papain-catalyzed aldol reaction a.
Entry Papain loading (U)
Yield (%)b
Ee (%)c
29
1
2
3
4
32
61
86
64
30
96
98
29
128
98
29
a The reaction conditions were as follows: trifluoromethyl ketone (0.6 mmol), acetone
(6.0 mmol), papain (32-128 U), DMF (0.95 mL), deionized water (0.05 mL) at 25 °C
for 72 h.
b Yield refers to the isolated yield.
c Ee was determined by HPLC analysis using a chiral column (OD-H).

Table 5. Effect of temperature on the papain-catalyzed aldol reaction a.
Entry T ( °C )
Yield (%)b
Ee (%)c
3
1
2
3
4
5
6
7
8
10
25
54
78
99
99
98
97
96
5
15
17
20
23
25
29
30
30
35
29
40
30
50
29
9
30 (no enzyme)
--
a
The reactions conditions were as follows: trifluoromethyl ketone (0.6 mmol),
acetone (6.0 mmol), papain (96 U), DMF(1.19 mL) , deionized water (0.06 mL) at
10-50 °C for 72 h.
b Yield refers to the isolated yield.
c Ee was determined by HPLC analysis using a chiral column (OD-H).

Table 6. Substrate scope of the papain-catalyzed aldol reaction a.
Entry
1
1
2
No. Product
Time (h)
48
Yield (%)b
97
3a
2
3
3b
3c
3d
48
48
48
90
95
96
4
5
3e
3f
48
48
72
94
97
42
6
7c
3g

8
3h
3i
48
48
48
48
72
72
72
72
72
72
95
96
99
92
70
72
75
56
58
67
9
10
11
12d
3j
3k
3l
3m
3n
3o
3p
3q
13c
14c
15c
16c
17c
a
Unless otherwise noted, reaction conditions: a mixture of 1 (0.6 mmol), 2 (6.0
mmol), papain (96 U), DMF (1.19 mL), deionized water (0.06 mL) at 30 °C.
b Yield refers to the isolated yield.

c Papain loading of 192 U.
d
Papain loading of 192 U, dr (52:48) was determined by 1H NMR and HPLC (chiral
column AS-H).

Table 7. Kinetic parameters for the papain-catalyzed aldol reaction a.
Entr
Substrate
KM (M)
kcat (h-1)
kcat/KM (M-1 h-1)
y
1b
Trifluoromethyl ketone 0.102
(1a)
Acetone (2a)
98.5
965.7
2c
0.051
65.0
1274.5
a The reactions were carried out in DMF (0.24 mL) and deionized water (0.01 mL) at
30 °C for 1 h; the kinetic parameters were obtained at the enzyme concentration of
1.04 mM. The experiments were based on HPLC determination of the product.
b The concentration of 1a varied from 0.10 M to 0.58 M, with 2a (4.80 M).
c The concentration of 2a varied from 0.12 M to 0.72 M, with 1a (0.48 M).